Comparison of in Vitro Antibacterial Activity of Epaltes Divaricata and Vetiveria Zizanioides Against Methicillin- Resistant Staphylococcus Aureus

Hindawi Scientiﬁca Volume 2020, Article ID 8239053, 8 pages https://doi.org/10.1155/2020/8239053

Research Article Comparison of In Vitro Antibacterial Activity of Epaltes divaricata and Vetiveria zizanioides against Methicillin- Resistant Staphylococcus aureus

Hasanga Rathnayake,1 Manikkuwadura Hasara Nethmini De Zoysa,2 Ruwani Punyakanthi Hewawasam ,1 and Weerasinghe Mudiyanselage Dilip Gaya Bandara Wijayaratne 3

1Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Matara, Sri Lanka 2Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Ruhuna, Matara, Sri Lanka 3Department of Microbiology, Faculty of Medicine, University of Ruhuna, Matara, Sri Lanka

Correspondence should be addressed to Ruwani Punyakanthi Hewawasam; [email protected]

Received 29 May 2020; Accepted 25 June 2020; Published 14 July 2020

Academic Editor: Katsumi Doi

Copyright © 2020 Hasanga Rathnayake et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital- and community-acquired infections worldwide. )erefore, this study was conducted to explore the antibacterial activity of the two medicinal plants Epaltes divaricata and Vetiveria zizanioides against strains of MRSA which were isolated from patients with skin and soft tissue infections. Hexane, ethanol, and water extracts of E. divaricata (whole plant) and V. zizanioides (roots) were prepared. Clinical isolates of MRSA strains (n � 20) were used for the study. Bacterial susceptibility was tested using a disc diffusion assay. Minimum inhibitory concentration (MIC) was determined by a broth microdilution method. Vancomycin was used as the positive control. Hexane, ethanol, and water extracts of E. divaricata showed inhibitory zones against MRSA. Except for water extract, both hexane and ethanol extracts of V. zizanioides showed inhibitory zones. MIC ranges of hexane, ethanol, and water extracts in E. divaricata were 0.012–0.32 mg/mL, 0.019–2.4 mg/mL, and 0.019–0.48 mg/mL, respectively. Respective MIC ranges of hexane and ethanol extracts of V. zizanioides were 0.003–0.032 mg/mL and 0.019–2.4 mg/mL. )e hexane extract of V. zizanioides inhibited 55% of the selected MRSA strains at a relatively low MIC value of 0.012 mg/mL. )e hexane extract of both plants demonstrated inhibition of 75% of MRSA strains at a MIC value of 0.064 mg/mL. Ethanol extract of V. zizanioides and E. divaricata, respectively, inhibited 70% and 45% of MRSA strains at the MIC of 0.096 mg/mL, whereas water extract of E. divaricata inhibited 80% of MRSA strains at the same MIC. Both E. divaricata and V. zizanioides were equally effective against MRSA at a MIC of 0.064 mg/mL. But V. zizanioides was more effective since the hexane extract inhibited more than 50% of MRSA strains at significantly a lower MIC value of 0.012 mg/ mL. Fractionation, purification, and identification of active compounds will warrant further evaluation of the therapeutic potential of both plant extracts.

1. Introduction antibiotic-resistant organisms, methicillin-resistant Staph- ylococcus aureus (MRSA) causes a plethora of diseases in- Antimicrobial resistance has become a major public health volving the skin, soft tissue, bone, and joints [2]. It is one of issue in the 21st century which has threatened the prevention the major causes of the hospital- and community-acquired and the treatment of a wide variety of infections caused by infections [2]. )e resistance for β-lactam antibiotics in bacteria, viruses, and fungi. )e rate of emergence of an- MRSA is acquired by the expression of mecA gene [3]. tibiotic-resistant human pathogens is greater compared to Majority of clinical isolates of MRSA are also resistant to the discovery of new antibiotic drugs [1]. Among the clindamycin, ciprofloxacin, and levofloxacin [4]. 2 Scientifica

MRSA infections are prevalent in Sri Lanka mainly due dimethyl sulfoxide (DMSO) separately, and the final con- to prophylactic and empiric use of antibiotics [5]. It is highly centration of each extract was adjusted to 300 mg/ml. Each prevalent not only in hospitals but also in community extract was stored at 4°C in sterile airtight containers for samples in Sri Lanka [6]. Vancomycin is still the most further studies. commonly used antibiotic for multidrug-resistant S. aureus, but recent studies report the occurrence of S. aureus strains that are resistant to vancomycin in many countries including 2.2.2. Water Extract. 2.62 g of dried plant material was Sri Lanka [7]. )erefore, an urge of the discovery of new refluxed in 60 ml of distilled water for 3 hrs to obtain the drugs to combat drug-resistant microorganisms including S. water extract. )e resulting crude extract was dissolved in a aureus is essential. minimum amount of 10% DMSO, and the final concen- Medicinal plants are a key source of alternative medicine tration of the extract was adjusted to 300 mg/ml. )e extract ° for fighting against diseases since ancient times. )ey are was stored at 4 C in sterile airtight containers for further rich sources of valuable secondary metabolites which are studies. capable of inhibiting microorganisms [8]. )ese natural products are also promising sources for the synthesis of novel antibacterial compounds. )e whole plant of Epaltes 2.3. Test Organisms. MRSA strains (n � 20) were used for divaricata and roots of Vetiveria zizanioides (Figure 1) are the study along with CLSI (Clinical and Laboratory widely used in traditional medicine in Sri Lanka [9]. Veti- Standards Institute) standard strain of methicillin-sen- veria zizanioides (“Sevendara” in Sinhala) is commonly sitive S. aureus (MSSA-ATCC 25923) as the reference found in the warmer parts of the island. It is being used in organism. )e MRSA strains used in this study were Ayurveda for the treatment of typhoid fever, haemoptysis, isolated from pus samples obtained for culture and sen- phthisis, anaemia, skin and blood diseases, urinary disor- sitivity testing from patients having skin and soft tissue ders, piles, oedema, anorexia, chronic dyspepsia, flatulence, infections among patients admitted to Teaching Hospital, and acute and chronic congestion of the liver and jaundice Karapitiya, Sri Lanka. Organisms were subcultured for 24 [10]. Epaltes divaricata (“Heen-mudamahana” in Sinhala) is hours on blood agar and MacConkey agar plates and being used to treat skin diseases, oedema, pains, headaches, confirmed by gram stain, catalase test, slide and tube epilepsy, jaundice, haemorrhoids, hernia, and dysuria [11]. coagulase tests, and the zone diameter of cefoxitin (30 μg) One of our previous studies reported the antibacterial on Mueller–Hinton agar at the Department of Microbi- activity of E. divaricata and V. zizanioides against standard ology, Faculty of Medicine, University of Ruhuna, Sri organisms such as Staphylococcus aureus (ATCC 25923), Lanka. Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 700603) [12]. However, in vitro studies on these plants for multidrug- 2.4. Bacterial Susceptibility Testing. Bacterial susceptibility resistant organisms are not available. )erefore, this study was tested using the disc diffusion assay. Crude extract, explores the antibacterial activity against MRSA strains 10-fold, and 100-fold dilutions of each plant extract were which were isolated from pus samples obtained for culture prepared in 10% DMSO. Bacteria cell suspensions were and sensitivity testing from patients having skin and soft adjusted to 0.5 McFarland turbidity standards to prepare × 8 tissue infections. 1 10 CFU/ml inoculum. Each standardized inoculum was introduced and evenly distributed on the surface of 2. Methods sterile Mueller–Hinton agar plates. Previously prepared sterile filter paper discs (Whatman No. 1, diame- 2.1. Plant Collection and Authentication. Whole plants of ter � 6 mm) were soaked in 10 μL of each plant extract. Epaltes divaricata and roots of Vetiveria zizanioides (Fig- )ey were placed on the seeded Mueller–Hinton agar ure 1) were collected from the Southern Province in Sri plates. Vancomycin (30 μg/disc) disk was used as the Lanka. Authentication of the plants was confirmed at the positive control, and 10% DMSO-soaked filter paper disk National Herbarium, Botanical Gardens, Peradeniya, Sri was used as the negative control. )e same procedure was Lanka. )e plant material was thoroughly washed and dried used for all the MRSA strains used. )e plates were in- in a hot air oven at 40°C for 3-4 days. Dried plant material cubated aerobically at 35 ± 2°C for 18–24 hr. After incu- was ground to a coarse powder and stored in sterile airtight bation, diameters of the zones of inhibition were containers at 4°C. measured using a Vernier caliper. Each test was carried out in triplicate, and the average values of the diameters were considered. Activity index (AI) and relative per- 2.2. Preparation of Plant Extracts centage inhibition (RPI) for each extract were calculated 2.2.1. Ethanol and Hexane Extracts. 20 g of coarse powder of using the following formula [13]: each plant material was mixed with 200 ml of the solvent. 100(X − Y) RPI � , )e contents were placed in a mechanical shaker for 72 hr at (Z − Y) 25°C. )en, the extracts were filtered, and the solvent was (1) inhibition zone diameter of the sample evaporated using a rotary evaporator. )e resulting crude AI � , extracts were dissolved in a minimum amount of 10% inhibition zone of the standard Scientifica 3

(a) (b)

Figure 1: (a) Botanical name: Epaltes divaricata, local name: “Heen mudamahana,” family: Compositae; (b) botanical name: Vetiveria zizanioides, local name: “Sevendara,” family: Gramineae. where X � total area of inhibition of the test extract, Y � total 3. Results area of inhibition of the solvent, and Z � total area of inhibition of the standard drug. Disc diffusion assay was performed to detect the presence of )e total area of the inhibition was calculated by using inhibitory zones of E. divaricata and V. zizanioides against area � πr2; where r � radius of zone of inhibition. MRSA. Inhibitory zones were observed from crude extracts (hexane, ethanol, and water) of E. divaricata against MRSA (Figure 2 and Table 1). Except for water extract, both hexane 2.5. Determination of Minimum Inhibitory Concentration and ethanol extracts of V. zizanioides showed inhibitory (MIC). Broth microdilution method was used to determine zones (Figure 3). Relatively larger inhibitory zones ranging the MIC. Serial 5-fold dilutions of the plant extracts were from 13.1 to 18.7 mm were observed from the undiluted prepared in the 10% DMSO, yielding serial dilutions of the crude ethanol extract of E. divaricata, while diameters of crude extract. Bacterial inoculum was prepared in Muel- inhibitory zones of water and hexane extracts were lower ler–Hinton broth, and the turbidity was adjusted to ap- ranging from 6.6 mm to 15.6 mm and 6.7 mm to 13.1 mm, proximately 0.5 McFarland turbidity standards. 96-well respectively. Similarly, in V. zizanioides, larger inhibitory microtitre plates were used for the MIC assay, and 150 μL of zones were observed from undiluted ethanol extract ranging plant extract was added to each well of the microplate. 50 μL from 6.4 mm to 13.3 mm followed by hexane extract which of bacterial suspension was added to each well except the showed a diameter range of 6.5–11.4 mm. Highest AI and negative controls. Vancomycin (MIC ≤ 2 μg/ml) was used as RPI values were observed from crude ethanol extracts of positive control. 10% DMSO and plant extracts without both plants (Figure 4). Among diluted extracts of E. bacterial suspension were used as the negative controls. divaricata, inhibitory zones were observed from 10-fold to Microtiter plates were incubated at 35 ± 2°C for 24 hr. An- 100-fold diluted ethanol extracts. In V. zizanioides, only the timicrobial activity was assessed by the measurement of 10-fold diluted ethanol extract showed inhibitory zones. absorbance at 630 nm using a microplate reader. )e lowest Mean inhibitory zone of vancomycin (30 μg/disc) for MRSA concentration (highest dilution) of the extract that does not and MSSA was 20.6 mm and 20.8 mm, respectively. Com- produce bacterial growth depending on the absorbance was pared to the MRSA strains, MSSA (ATCC 25923) also regarded as MIC. )e assay was done in triplicate for each showed similar susceptibility pattern for the corresponding extract separately, and average absorbance values were used plant extracts (Table 1). to determine the MIC. MIC ranges of hexane, ethanol, and water extracts in E. divaricata were 0.012–0.32 mg/mL, 0.019–2.4 mg/mL, and 0.019–0.48 mg/mL, respectively. Respective MIC ranges of 2.6. Ethical Approval. Ethical approval was obtained from hexane and ethanol extracts of V. zizanioides were the Ethical Review Committee, Faculty of Medicine, Uni- 0.003–0.032 mg/mL and 0.019–2.4 mg/mL. )e hexane ex- versity of Ruhuna, Sri Lanka. tract of V. zizanioides inhibited 75% of the selected MRSA strains at relatively low MIC values ranging from 0.003 to 0.064 mg/mL. )e hexane extract of E. divaricata inhibited 2.7. Statistical Analysis. Mean standard error of means 75% of MRSA strains at MIC values ranging from 0.012 mg/ (SEM) was calculated to express data. )e data were analysed mL to 0.064 mg/mL. Ethanol extract of V. zizanioides using one-way ANOVA to compare the mean between inhibited 70% of MRSA strains at the MIC values ranging groups. Mean differences at each point were considered from 0.019 mg/mL to 0.096 mg/mL, whereas ethanol and significant at p < 0.05. water extracts of E. divaricata, respectively, inhibited 45% 4 Scientifica

P P P

N N C3 C3 N

C3 C2 C1 C1 C1 C2 C2

(a) (b) (c)

Figure 2: Inhibitory zones of crude extracts of E. divaricata: (a) hexane extract; (b) ethanol extract; (c) water extract. P: positive control; N: negative control; C1: crude extract; C2: 10-fold dilution of crude extract; C3: 100-fold dilution of crude extract.

Table 1: Diameter values of the disk diﬀusion assay of E. divaricata and V. zizanioides against MSSA and MRSA strains. Inhibition zone diameter (mm) Extract Concentration MRSA MSSA (ATCC 25923) E. divaricata V. zizanioides E. divaricata V. zizanioides 40 mg/mL (crude) 9.9 (6.7–13.1)∗, n � 20 8.3 (6.5–11.4), n � 20 13.7 ± 0.1# 11.4 ± 0.2 Hexane 4 mg/mL (10x dilution) — — — — 0.4 mg/mL (100x dilution) — — — — 300 mg/mL (crude) 15.4 (13.1–18.7), n � 20 8.7 (6.4–13.3), n � 8 16.3 ± 0.2 12.1 ± 0.2 Ethanol 30 mg/mL (10x dilution) 10.1 (7.4–11.7), n � 18 6.8 (6.3–9.0), n � 8 13.4 ± 0.2 7.3 ± 0.3 3 mg/mL (100x dilution) 7.1 (6.3–10.1), n � 8 — 7.6 ± 0.1 — 150 mg/mL (crude) 9.4 (6.6–15.6), n � 20 — 7.4 ± 0.2 — Aqueous 15 mg/mL (10x dilution) — — — — 1.5 mg/mL (100x dilution) — — — — Vancomycin 30 μg/disc 20.6 ± 2.2† 20.8 ± 0.1# n denotes the number of isolates that showed a measurable diameter; ∗data were expressed as mean (min-max); #data were expressed as mean ± SEM; †mean ± standard deviation.

P P

N N C3 C3

C1 C1 C2 C2

(a) (b)

Figure 3: Inhibitory zones of crude extracts of V. zizanioides: (a) hexane extract; (b) ethanol extract. P: positive control; N: negative control; C1: crude extract; C2: 10-fold dilution of crude extract; C3: 100-fold dilution of crude extract. and 80% of MRSA strains at the same MIC range. Relatively E. divaricata was needed to inhibit 20% of selected MRSA high MIC values of ethanol extracts of E. divaricata and V. strains (Tables 2 and 3). zizanioides ranging from 0.48 to 2.4 mg/mL were needed to MRSA which were not effectively inhibited by ex- inhibit 55% and 30% of selected MRSA strains, respectively. tracts of E. divaricata was effectively inhibited by extracts Similarly, 0.48 mg/mL concentration of the water extract of of V. zizanioides, and vice versa. MRSA nos. 2 and 3 were Scientifica 5

0.9 60 0.8 50 0.7 0.6 40 0.5 30 0.4 0.3 20 Activity index Activity 0.2 10 0.1 Relative percentage inhibition 0 0 ED (MRSA) VZ (MRSA) ED (MSSA) VZ (MSSA) ED (MRSA) VZ (MRSA) ED (MSSA) VZ (MSSA) Plant extracts Plant extracts Hexane ED; E. divaricata Hexane ED; E. divaricata Ethanol VZ; V. zizanioides Ethanol VZ; V. zizanioides Aqueous Aqueous

(a) (b)

Figure 4: Activity index (a) and relative percentage inhibition (b) of crude extracts of E. divaricata and V. zizanioides for MRSA and MSSA.

Table 2: MIC values of E. divaricata and V. zizanioides against MRSA strains. Minimum inhibitory concentration (mg/mL) MRSA strain Hexane extract Ethanol extract Water extract E. divaricata V. zizanioides E. divaricata V. zizanioides E. divaricata 1 0.064 0.064 0.09 0.48 0.09 2 0.064 0.012 2.40 0.09 0.09 3 0.32 0.012 2.40 0.09 0.48 4 0.32 0.32 0.09 0.09 0.48 5 0.32 0.012 0.48 0.09 0.09 6 0.32 0.012 0.48 048 0.09 7 0.064 0.003 0.09 0.09 0.09 8 0.32 0.064 0.48 2.40 0.48 9 0.012 0.32 0.48 2.40 0.48 10 0.012 0.32 2.40 0.48 0.09 11 0.064 0.012 0.48 0.09 0.09 12 0.012 0.012 0.019 0.019 0.09 13 0.064 0.003 0.09 0.09 0.09 14 0.064 0.064 0.48 0.09 0.09 15 0.064 0.012 0.09 0.019 0.09 16 0.064 0.32 0.09 0.09 0.019 17 0.064 0.012 0.09 0.019 0.09 18 0.012 0.32 0.48 0.09 0.09 19 0.012 0.064 0.48 0.09 0.019 20 0.064 0.012 0.09 0.48 0.09 Mean (SEM) n � 20 0.064∗ (0.012–0.32) 0.098∗ (0.003–0.32) 0.480∗ (0.019–2.4) 0.388∗ (0.019–2.4) 0.399∗ (0.019–0.48) MSSA# 1.6 0.003 0.48 2.4 1.2 ∗Data were expressed as mean (min-max); #methicillin-sensitive S. aureus (ATCC 25923). inhibited at MIC of 2.4 mg/mL of the ethanol extract of E. 2.4 mg/mL of ethanol extract, but it was also inhibited at divaricata, but they were inhibited at 0.012 mg/mL by the MIC of 0.012 mg/mL of hexane extract of E. divaricata. hexane extract of V. zizanioides. MRSA nos. 9 and 10 that )e reference strain of S. aureus (ATCC 25923) was more were inhibited at MIC of 0.32 mg/mL of hexane extract of sensitive for the hexane extract of V. zizanioides which V. zizanioides were inhibited at 0.012 mg/mL by the required MIC of 0.003 mg/mL. Relatively high MIC hexane extract of E. divaricata. Furthermore, MIC values values ranging from 0.48 to 2.4 mg/mL of ethanol and obtained for the different extracts of the same plant for hexane extracts were needed to inhibit S. aureus (ATCC individual MRSA also varied. MRSA no. 6 which was 25923) (Table 2). )ere were no statistically inhibited at MIC of 0.48 mg/mL of ethanol extract was significant differences in MIC between hexane, ethanol, also inhibited at MIC of 0.012 mg/mL of hexane extract of and aqueous extracts of E. divaricata and V. zizanioides V. zizanioides. MRSA no. 9 was inhibited at MIC of (p > 0.05). 6 Scientifica

Table 3: Percentage of MRSA strains inhibited at diﬀerent MIC values of E. divaricata and V. zizanioides. Percentage (%) of MRSA inhibited MIC (mg/mL) Hexane extract Ethanol extract Water extract E. divaricata V. zizanioides E. divaricata V. zizanioides E. divaricata 0.003 — 10% (n � 2) — — — 0.012 25% (n � 5) 45% (n � 9) — — — 0.019 — — 5% (n � 1) 15% (n � 3) 10% (n � 2) 0.064 50% (n � 10) 20% (n � 4) — — — 0.096 — — 40% (n � 8) 55% (n � 11) 70% (n � 14) 0.320 25% (n � 5) 25% (n � 5) — — — 0.480 — — 40% (n � 8) 20% (n � 4) 20% (n � 4) 2.400 — — 15% (n � 3) 10% (n � 2) — Total 100% (n � 20) 100% (n � 20) 100% (n � 20) 100% (n � 20) 100% (n � 20) n � number of MRSA strains.

4. Discussion activities of E. divaricata in 2015, but they have used a single strain of MRSA, and MIC was not reported [16]. Furthermore, )e results revealed that the crude extracts of E. divaricata their study revealed the presence of phenolic compounds, 2- and V. zizanioides were effective against the MRSA strains as butenamide, N-(4-fluorophenyl)–methyl trans-cinnamyl observed by the zones of inhibition. Disc diffusion assay was tiglate silane, trichlorocyclohexyl silane, and its derivatives in E. performed as a preliminary screening to confirm the in- divaricata, which could be responsible for the antibacterial hibitory effects of the plant extracts against MRSA. Highest activity. Sivagurunathan and Krishnamoorthy reported the AI and RPI values were obtained from crude ethanol extracts antibacterial properties of V. zizanioides for a single strain of of the plants. Since undiluted crude extracts were more MRSA in 2017, but MIC was not reported. High concentra- effective against MRSA, it is assumed that the antibacterial tions of phenolic compounds were identified in the ethanol effect may be due to the presence of possible active ingre- extract, and HPLC results showed the presence of different dients in high concentrations. However, the strength of an types of unidentified phytoconstituents [17]. Zuo et al. reported inhibitory effect cannot be compared by the results of disc anti-MRSA activity of 19 medicinal plants against 9 MRSA diffusion assay due to the varying concentrations of the strains (except E. divaricata and V. zizanioides) with MIC crude extracts. )erefore, MIC was determined as the range of 1.25–3.07 mg/ml [18]. standard method to compare the antibacterial activity of the )ere were many reports in the recent past on the an- plant extracts. timicrobial activity of medicinal plants against many mi- )e MIC values obtained for the extracts against the croorganisms including MRSA. Dahiya and Purkayastha MRSA varied from one plant extract to another. One reason reported antibacterial activity of seven medicinal plants for a would be the extraction of different constituents in partic- single strain of MRSA in 2012 [19]. Extracts of Tulsi, ular solvents depending on their chemical and physical oregano, rosemary, and Aloe vera were included in their properties especially the polarity. Furthermore, due to ge- study, and MIC values were found to be in the range of netic variability, different MRSA could have acted differently 1.56–6.25 mg/ml. When compared to other studies con- with chemicals in each extract. Organisms which were not ducted by Okwu et al., Zuo et al., and Dahiya and Pur- effectively inhibited by E. divaricata were effectively kayastha [18–20], extracts of E. divaricata and V. zizanioides inhibited by extract of V. zizanioides, and vice versa, sug- in the present study exhibited relatively lower MICs against gesting the effectiveness of both plants towards different MRSA. strains of MRSA. However, V. zizanioides is more effective Trong Le et al. reported antimicrobial activities of es- since hexane extracts inhibited majority of MRSA strains at sential oils extracted from Paramignya trimera and Lim- relatively lower MIC such as 0.003 mg/mL. Low polar active nocitrus littoralis which were used in the Vietnamese compounds present in the hexane extract may be responsible traditional medicine [21]. P. trimera strongly inhibited S. for this antimicrobial activity. As observed in our previous aureus ATCC 43300 and the S. aureus clinical strain with studies, compared to the MRSA strains, S. aureus ATCC MIC and MLC (minimum lethal concentration) values of 2% standard (ATCC 25923) also showed similar susceptibility (v/v), but L. littoralis did not inhibit S. aureus. Oil extracted pattern for both plants [12]. )is could be due to the genetic from P. trimera consisted of β-caryophyllene, β-car- similarities between MSSA (ATCC 25923) and MRSA. yophyllene oxide, 7-epi-α-eudesmol, and c-muurolene as Studies conducted in Sri Lanka revealed that certain me- major components. )is suggested that the presence of dicinal plants which are being used in traditional medicine β-caryophyllene and β-caryophyllene oxide may be re- possess anti-MRSA properties [14, 15]. However, local studies sponsible for the antibacterial activity of P. trimera against S. on E. divaricata and V. zizanioides, related to MRSA, were aureus. Another Vietnamese study on essential oil extracted deficient. On the contrary, very few studies were available from from the leaves of Leoheo domatiophorus inhibited S. aureus other countries on these plants which reported anti-MRSA ATCC 43300 and the S. aureus clinical strain [22]. Both S. activity. Glorybai et al. reported the presence of antibacterial aureus strains were inhibited at MIC of 0.25% (v/v) and Scientifica 7

MLC of 0.5% (v/v). GC/MS (gas chromatography/mass toxicological studies on hexane and ethanol extracts have spectrometry) analysis of the essential oil of L. domatio- not been conducted yet. To develop potential therapeutics phorus indicated the presence of sesquiterpene hydrocar- with anti-MRSA properties from these plant extracts, the bons, oxygenated sesquiterpenes as the main classes of toxicity of active compounds and their pharmacokinetics compounds. Viridiflorene, (−)-δ-cadinene, and c-muur- should be investigated. In vivo studies including clinical olene were present at higher concentrations, while trials are necessary to confirm the efficacy of active α-muurolene, c-cadinene, (+)-aromadendrene, α-cadinol, compounds. and globulol were present at lower concentrations. Many other studies published recently also reported the 5. Conclusion and Recommendation presence of compounds of natural origin that could alleviate the symptoms associated with skin infections. A study Here, we present preliminary findings of antibacterial ac- conducted on water extract of Borojoa patinoi which was tivities of crude extracts of E. divaricata and V. zizanioides used in Colombian traditional medicine did not inhibit S. against twenty clinical isolates of MRSA. Both plants are aureus M121 which is resistant to both methicillin and equally effective against MRSA at a MIC of 0.064 mg/mL, but vancomycin [23]. However, the extract inhibited multidrug- V. zizanioides is more effective since the hexane extract resistant (MDR) Pseudomonas aeruginosa and the UHPLC inhibited more than 50% of MRSA strains at significantly (ultrahigh performance liquid chromatography) analysis of lower MIC of 0.012 mg/mL. Presence of phenolic com- the plant extract showed the presence of 26 phenolic pounds, alkaloids, and flavonoids identified in a previous compounds including hydroxycinnamic acids, phenolic study confirmed the presence of possible active compounds acids, flavonols, flavan-3-ols, flavonones, flanones, tyrosol responsible for the antimicrobial effect in both E. divaricata ester, and dihydrochalcones. Mazzarello et al. reported that a and V. zizanioides. Since hexane extract was proven as the skin cream containing propolis 20%, tea tree oil 3%, and Aloe most active extract, low polar active phytochemicals present vera 10% was effective in reducing acne [24]. Pathogenesis of in the plant extracts may be responsible for this antimi- acne is associated with Propionibacterium acnes, S. aureus, crobial activity. Fractionation, purification, and identifica- and S. epidermidis. Propolis consists of flavonoids, caffeic tion of active compounds are essential in developing these acid, benzoic acid, and cinnamic acid. Tea tree oil contains extracts as potential therapeutics. In vivo studies will be terpinen-4-ol, which shows an important role in the anti- performed with purified active components to investigate microbial activity. Besides, another study on acne reported their pharmacokinetics and toxic effects in the future. antiacne efficacy of two essential oils extracted from Orig- anum vulgare and Myrtus communis L. [25]. Presence of Data Availability monoterpene, diterpene, sesquiterpene hydrocarbons, azu- )e data used to support the findings of the present study are lene, alcohols, aldehydes, and ketones in essential oils may available from the corresponding author upon request. have attributed to the antibacterial effects against MDR bacteria [26]. One of our previous studies also confirmed the presence Conflicts of Interest of tannins, phenolic compounds, cardiac glycosides, flavo- )e authors declare that they have no conflicts of interest. noids, alkaloids, and saponins in both E. divaricata and V. zizanioides plant extracts [12]. Dos Santos et al. reported that Authors’ Contributions β-vetivenene, khusimol, vetiselinenol, isovalencenol, veti- venic acid, α-vetivone, and β-vetivone are the major con- Hasanga Rathnayake and Manikkuwadura Hasara Nethmini stituents of dichloromethane fraction of Vetiveria De Zoysa contributed equally to this work. zizanioides [27]. Antimicrobial activity of plant phenolics including flavonoids has also been documented in the past Acknowledgments [28]. As evident from the abovementioned studies, active compounds isolated from medicinal plants have a significant )is study was funded by the Faculty Research Grant of the potential to alleviate the symptoms associated with skin Faculty of Medicine, University of Ruhuna, Sri Lanka. )e infections. Composition of active compounds in these plants authors are grateful to the technical officers and other could vary from one country to another due to different nonacademic staff members at the Departments of Bio- geographical and environmental conditions. Antimicrobial chemistry, Microbiology, and Nuclear Medicine Unit, activities could be enhanced if the active compounds of the Faculty of Medicine, University of Ruhuna, Sri Lanka, for species under study that were collected from Sri Lanka are their technical and other assistance to make the present purified [29]. )erefore, further investigations which involve study a success. fractionation, purification, and identification of active compounds are necessary to develop these two plants as References potential therapeutic agents in the future. Previous studies [1] S. B. Zaman, M. A. Hussain, R. Nye, V. Mehta, K. T. Mamun, conducted in our laboratory reported the absence of any and N. Hossain, “A review on antibiotic resistance: alarm bells toxicological effects (biochemical, haematological, and his- are ringing,” Cureus, vol. 9, no. 6, Article ID e1403, 2017. topathological) in ICR mice when treated with water extracts [2] A. Hassoun, P. K. Linden, and B. Friedman, “Incidence, of E. divaricata and V. zizanioides [9]. However, prevalence, and management of MRSA bacteremia across 8 Scientifica

patient populations—a review of recent developments in [16] L. Glorybai, K. B. Kannan, M. V. Arasu, N. A. Al-Dhabi, and MRSA management and treatment,” Critical Care (London, P. Agastian, “Some biological activities of Epaltes divaricata England), vol. 21, no. 1, p. 211, 2017. L.—an in vitro study,” Annals of Clinical Microbiology and [3] J. Rolo, P. Worning, J. Boye Nielsen, and R. Sobral, “Evidence Antimicrobials, vol. 14, p. 18, 2015. for the evolutionary steps leading to mecA-mediated beta- [17] S. K. Sivagurunathan and G. Krishnamoorthy, “Comparative lactam resistance in staphylococci,” PLoS Genetics, vol. 13, study on high-performance thin layer chromatography profile Article ID e1006674, 2017. and antimicrobial activity of ethanolic and hydroalcoholic [4] A. C. Gales, H. S. Sader, J. Ribeiro, C. Zoccoli, A. Barth, and extract of Vetiveria zizanioides L. root,” Asian Journal of A. C. Pignatari, “Antimicrobial susceptibility of gram-positive Pharmaceutical and Clinical Research, vol. 10, no. 6, bacteria isolated in Brazilian hospitals participating in the pp. 336–339, 2017. SENTRY program (2005–2008),” Brazilian Journal of Infec- [18] G. Y. Zuo, G. C. Wang, Y. B. Zhao et al., “Screening of Chinese tious Diseases, vol. 13, no. 2, pp. 90–98, 2009. medicinal plants for inhibition against clinical isolates of [5] E. Corea, T. de Silva, and J. Perera, “Methicillin-resistant methicillin-resistant Staphylococcus aureus (MRSA),” Journal Staphylococcus aureus: prevalence, incidence and risk factors of Ethnopharmacology, vol. 120, no. 2, pp. 287–290, 2008. associated with colonization in Sri Lanka,” Journal of Hospital [19] P. Dahiya and S. Purkayastha, “Phytochemical screening and Infection, vol. 55, no. 2, pp. 145–148, 2003. antimicrobial activity of some medicinal plants against multi- [6] W. Samaranayake, L. Karunanayake, and C. Patabendige, drug resistant bacteria from clinical isolates,” Indian Journal “Characteristics of community acquired and hospital acquired of Pharmaceutical Sciences, vol. 74, no. 5, pp. 443–450, 2012. [20] M. U. Okwu, M. Olley, A. O. Akpoka, and O. E. Izevbuwa, methicillin resistant Staphylococcus aureus isolates in the “Methicillin-resistant Staphylococcus aureus (MRSA) and national hospital of Sri Lanka,” Sri Lankan Journal of Infec- anti-MRSA activities of extracts of some medicinal plants: a tious Diseases, vol. 9, no. 1, pp. 24–31, 2019. brief review,” AIMS Microbiology, vol. 5, no. 2, pp. 117–137, [7] D. R. Chung, C. Lee, Y. R. Kang et al., “Genotype-specific 2019. prevalence of heterogeneous vancomycin-intermediate [21] N. Trong Le, D. Viet Ho, T. Quoc Doan et al., “Biological Staphylococcus aureus in Asian countries,” International activities of essential oils from leaves of Paramignya trimera Journal of Antimicrobial Agents, vol. 46, no. 3, pp. 338–341, (oliv.) Guillaum and Limnocitrus littoralis (Miq.) Swingle,” 2015. Antibiotics, vol. 9, no. 4, p. 207, 2020. [8] R. Subramani, M. Narayanasamy, and K. D. Feussner, “Plant- [22] N. Trong Le, D. Viet Ho, T. Quoc Doan et al., “In vitro derived antimicrobials to fight against multi-drug-resistant antimicrobial activity of essential oil extracted from leaves of human pathogens,” 3 Biotech, vol. 7, p. 172, 2017. Leoheo domatiophorus Chaowasku, D. T. Ngo and H. T. Le in [9] R. P. Hewawasam, K. A. P. W. Jayatilaka, L. K. B. Mudduwa, Vietnam,” Plants (Basel, Switzerland), vol. 9, no. 4, p. 453, and C. Pathirana, “Toxicological evaluation of five Sri Lankan 2020. medicinal plants: a biochemical, haematological and histo- [23] C. Chaves-López,D. Usai, M. G. Donadu et al., “Potential of pathological assessment,” International Journal of Pharma- Borojoa patinoi Cuatrecasas water extract to inhibit noso- ceutical Sciences and Research, vol. 7, no. 10, pp. 4014–4021, comial antibiotic resistant bacteria and cancer cell prolifer- 2016. ation in vitro,” Food & Function, vol. 9, no. 5, pp. 2725–2734, [10] D. M. A. Jayaweera, Medicinal Plants (Indigenous and Exotic) 2018. used in Ceylon, Part III, National Science Council, Colombo, [24] V. Mazzarello, M. Donadu, M. Ferrari et al., “Treatment of Sri Lanka, 1982. acne with a combination of propolis, tea tree oil, and Aloe vera [11] M. D. Dassanayake and F. R. Fosberg, A Revised Handbook to compared to erythromycin cream: two double-blind inves- the Flora of Ceylon, Vol. 1–14, Amerind Publishers, New tigations,” Clinical Pharmacology: Advances and Applications, Delhi, India, 1980. vol. 10, pp. 175–181, 2018. [12] M. H. N. De Zoysa, H. Rathnayake, R. P. Hewawasam, and [25] V. Mazzarello, E. Gavini, M. G. Donadu et al., “Clinical as- W. M. D. G. B. Wijayaratne, “Determination of in vitro sessment of new topical cream containing two essential oils antimicrobial activity of five Sri Lankan medicinal plants combined with tretinoin in the treatment of acne,” Clinical, against selected human pathogenic bacteria,” International Cosmetic and Investigational Dermatology, vol. 13, pp. 233– Journal of Microbiology, vol. 2019, Article ID 7431439, 8 pages, 239, 2020. 2019. [26] S. Cannas, D. Usai, A. Pinna et al., “Essential oils in ocular [13] D. Dharajiya, P. Patel, M. Patel, and N. Moitra, “In vitro pathology: an experimental study,” Fe Journal of Infection in antimicrobial activity and qualitative phytochemical analysis Developing Countries, vol. 9, no. 6, pp. 650–654, 2015. of Withania somnifera (L.) dunal extracts,” International [27] D. S. Dos Santos, J. V. Oberger, R. Niero et al., “Seasonal Journal of Pharmaceutical Sciences Review and Research, phytochemical study and antimicrobial potential of Vetiveria vol. 27, no. 60, pp. 349–354, 2014. zizanioides roots,” Acta Pharmaceutica, vol. 64, no. 4, [14] T. Gunasekara, N. Radhika, K. Ragunathan et al., “Deter- pp. 495–501, 2014. mination of antimicrobial potential of five herbs used in [28] T. Hatano, H. Uebayashi, H. Ito, S. Shiota, T. Tsuchiya, and ayurveda practices against Candida albicans, Candida para- T. Yoshida, “Phenolic constituents of Cassia seeds and an- psilosis and methicillin resistant Staphylococcus aureus,” tibacterial effect of some naphthalenes and anthraquinones on Ancient Science of Life, vol. 36, no. 4, pp. 187–190, 2017. methicillin-resistant Staphylococcus aureus,” Chemical & [15] M. P. J. Dharmaratne, A. Manoraj, V. )evanesam et al., Pharmaceutical Bulletin, vol. 47, no. 8, pp. 1121–1127, 1999. [29] D. C. M. Costa, M. M. B Azevedo, D. O. E. Silva et al., “In vitro “Terminalia bellirica fruit extracts: in-vitro antibacterial ac- anti-MRSA activity of Couroupita guianensis extract and its tivity against selected multidrug-resistant bacteria, radical component Tryptanthrin,” Natural Product Research, vol. 31, scavenging activity and cytotoxicity study on BHK-21 cells,” no. 17, pp. 2077–2080, 2017. BMC Complementary and Alternative, vol. 18, no. 1, p. 325, 2018.